Fluorescence-coded mid-infrared photothermal microscope

ABSTRACT

Microscopic analysis of a sample includes a fluorescent dye disposed within the sample. A mid-IR optical source generates a mid-infrared beam, which is directed onto the sample to induce a temperature change by absorption of the mid-infrared beam. An optical source generates a probe beam directed to impinge on the sample. A detector detects fluorescent emissions from the sample when the probe beam impinges on the sample. A data acquisition and processing system acquires and processes the detected fluorescent emissions from the sample to: (i) generate a signal indicative of infrared absorption by the sample, (ii) generate a signal indicative of temperature in the sample based on the signal indicative of infrared absorption by the sample, (iii) generate an image of the sample using the signal indicative of temperature in the sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is related to and claims the benefit of U.S.Provisional Application No. 63/074,668, filed on Sep. 4, 2020, theentire contents of which are incorporated herein by reference.

BACKGROUND 1. Technical Field

The present disclosure is related to mid_infrared (IR) photothermal(MIP) imaging and, in particular, to a system and method for MIP imagingusing a thermally sensitive fluorescent dye to sense temperatureincrease in a sample induced by mid-infrared absorption.

2. Discussion of Related Art

MIP imaging is an emerging technique in which a beam of visible light isused to sense the photothermal lensing effect induced by infraredabsorption of molecules. This technology generally provides sub-micronspatial resolution defined by the visible probe beam. Yet, thephotothermal lensing effect is a weak effect for most materials. Forexample, the diffraction coefficient of poly (methyl methacrylate)(PMMA) changes approximately 0.1% per Kelvin. With such low sensitivityto temperature variation, measurements with high spatial resolutioncannot be obtained.

SUMMARY

According to one aspect, a system for microscopic analysis of a sampleis provided. A fluorescent dye is disposed within the sample. Amid-infrared (IR) optical source generates a mid-infrared beam, themid-infrared beam being directed onto at least a portion of the sampleto induce a temperature change in the portion of the sample byabsorption of the mid-infrared beam. An optical source generates a probebeam, the probe beam being directed to impinge on the sample. A detectordetects fluorescent emissions from the sample when the probe beamimpinges on the sample. A data acquisition and processing systemacquires and processes the detected fluorescent emissions from thesample to: (i) generate a signal indicative of infrared absorption bythe portion of the sample, (ii) generate a signal indicative oftemperature in the portion of the sample based on the signal indicativeof infrared absorption by the portion of the sample, (iii) generate animage of the portion of the sample using the signal indicative oftemperature in the portion of the sample.

In some exemplary embodiments, the fluorescent dye comprises at leastone of rhodamine B, fluorescein, cy2, cy3, Nile red and greenfluorescent protein.

In some exemplary embodiments, the mid-infrared beam is apulse-modulated beam. A pulse repetition frequency of pulses in themid-infrared beam can be in a range of 1.0 to 1,000 kHz and cannominally be 100 kHz. An on-time of a pulse of the mid-infrared beam canbe in a range of 1.0 nanosecond to 1.0 millisecond and in someembodiments can nominally be between 50 and 1000 nanoseconds. A dutycycle of the mid-infrared beam can be in a range of 0.01% to 50% and insome embodiments can be between 1 and 10%.

In some exemplary embodiments, the mid-infrared beam is scanned over aplurality of portions of the sample such that the image is generated fora plurality of portions of the sample.

In some exemplary embodiments, the mid-infrared beam is directed onto aplurality of portions of the sample, and the detector comprises atwo-dimensional array of detectors such that the image is generated forthe plurality of portions of the sample. In some exemplary embodiments,the detector comprises a camera.

In some exemplary embodiments, the system further comprises an objectiveconfigured to focus the mid-infrared beam onto the sample.

According to another aspect, a method for microscopic analysis of asample is provided. The method includes introducing a fluorescent dyewithin the sample; directing a mid-infrared beam being onto at least aportion of the sample to induce a temperature change in the portion ofthe sample by absorption of the mid-infrared beam; directing a probebeam to impinge on the sample; detecting fluorescent emissions from thesample when the probe beam impinges on the sample; and receiving andprocessing the fluorescent emissions from the sample, the processingincluding: (i) generating a signal indicative of infrared absorption bythe portion of the sample, (ii) generating a signal indicative oftemperature in the portion of the sample based on the signal indicativeof infrared absorption by the portion of the sample, (iii) generating animage of the portion of the sample using the signal indicative oftemperature in the portion of the sample.

In some exemplary embodiments, the fluorescent dye comprises at leastone of rhodamine B, fluorescein, cy2, cy3, Nile red and greenfluorescent protein.

In some exemplary embodiments, the mid-infrared beam is apulse-modulated beam. A pulse repetition frequency of pulses in themid-infrared beam can be in a range of 1.0 to 1,000 kHz and cannominally be 100 kHz. An on-time of a pulse of the mid-infrared beam canbe in a range of 1.0 nanosecond to 1.0 millisecond and in someembodiments can nominally be between 50 and 1000 nanoseconds. A dutycycle of the mid-infrared beam can be in a range of 0.01% to 50% and insome embodiments can be between 1 and 10%.

In some exemplary embodiments, the mid-infrared beam is scanned over aplurality of portions of the sample such that the image is generated fora plurality of portions of the sample.

In some exemplary embodiments, the mid-infrared beam is directed onto aplurality of portions of the sample, and the detector comprises atwo-dimensional array of detectors such that the image is generated forthe plurality of portions of the sample. In some exemplary embodiments,the detector comprises a camera.

In some exemplary embodiments, the system further comprises an objectiveconfigured to focus the mid-infrared beam onto the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure is further described in the detailed descriptionwhich follows, in reference to the noted plurality of drawings by way ofnon-limiting examples of embodiments of the present disclosure, in whichlike reference numerals represent similar parts throughout the severalviews of the drawings.

FIG. 1A includes a schematic diagram illustrating operation of afluorescence-enhanced mid-infrared photothermal microscope, according tosome exemplary embodiments.

FIG. 1B, which is a schematic diagram illustrating molecular structurechange of Rhodamine B caused by heating.

FIG. 2A includes a schematic diagram of a point scanning FE-MIPmicroscope, according to some exemplary embodiments. FIG. 2B is aschematic diagram of the effects at the sample being analyzed. FIG. 2Cincludes a schematic functional block diagram illustrating componentsfor control and analysis in the FE-MIP microscope, according to someexemplary embodiments.

FIG. 3A includes a schematic diagram of a wide-field FE-MIP microscope,according to some exemplary embodiments. FIG. 3B includes a schematicfunctional block diagram illustrating components for control andanalysis in the FE-MIP microscope of FIG. 3A, according to someexemplary embodiments.

FIG. 4A illustrates the FE-MIP and FTIR spectra for DMSO. FIG. 4Billustrates the FE-MIP and FTIR spectra for water. FIGS. 4C and 4Dillustrate the fluorescence and FE-MIP images of 200 nm polystyrenebeads.

FIG. 5A is the fluorescence image obtained for S. aureus bacteria,according to some exemplary embodiments. FIGS. 5B-5D are FE-MIP imagesof S. aureus at the 1650 cm⁻¹, 1080 cm⁻¹, and 1750 cm⁻¹ IR pump laserwave numbers, corresponding to DNA, protein and lipid, respectively.FIG. 5E is a curve illustrating the FE-MIP spectrum and scattering MIPspectrum for S. aureus.

FIG. 6A is the fluorescence image obtained for S. aureus bacteria,according to some exemplary embodiments. FIGS. 6B-6D are FE-MIP imagesof S. aureus at the 1650 cm⁻¹, 1080 cm⁻¹, and 1750 cm⁻¹ IR pump laserwave numbers, corresponding to corresponding to protein amide I band,nucleic acid phosphate band, and off-resonance, respectively.

FIG. 7A includes a schematic diagram of a point scanning FE-MIPmicroscope, according to some exemplary embodiments. FIG. 7B illustratesF-MIP spectra of DMSO supplemented with various thermo-sensitivefluorescent dyes.

FIGS. 8A through 8G illustrate F-MIP imaging and fingerprinting singleS. aureus.

FIGS. 9A though 9I illustrate Sc-MIP and F-MIP images of living MiaPaca2cancer cells.

FIG. 10A illustrates temporal synchronization of the IR pump pulse,fluorescence excitation pulse, and camera exposure. FIGS. 10B and 10Cillustrate a wide field fluorescence image of Cy2-stained S. aureus withIR on and IR off, designated as hot and cold, respectively. FIG. 10Dillustrates wide field F-MIP at 1650 cm⁻¹, and FIG. 10E illustratesF-MIP at 1400 cm⁻¹ off resonance.

FIGS. 11A-11N illustrate performance comparison between wide fieldSC-MIP and wide field F-MIP systems.

FIGS. 12A-12F illustrate normalized F-MIPs images exhibiting a moreuniform distribution of the signal arising from proteins in regionslabeled by dyes.

DETAILED DESCRIPTION

According to the present disclosure, highly sensitive probes areutilized to improve the detection sensitivity in a MIP microscope.According to the technology of the disclosure, a fluorescence-enhancedmid-infrared photothermal (FE_MIP) microscope with high sensitivity isprovided. Generally, MIP microscopy uses a pump-probe approach in whicha mid-infrared light vibrationally excites a sample, and a visible lightprobes the resulting thermal effect. Instead of measuring the scatteringmodulated by mid-infrared absorption as is commonly done in conventionalMIP microscopy, according to the present disclosure, a thermallysensitive fluorescent dye is deployed in the sample as the probe, andthe modulated fluorescence intensity is measured. According to theexemplary embodiments, the modulated fluorescence intensity can bemeasured in both a confocal mode and a wide field mode. The result ishigh imaging sensitivity and component specificity through fluorescencelabeling.

Chemical imaging plays an increasingly important role in studyingbiological systems. It combines molecular spectroscopy withhigh-resolution spatial information to create quantitative images ofmolecular distributions. The many conventional chemical imaging toolsinclude stimulated Raman scattering microscopy, Fourier Transforminfrared (FTIR) spectroscopy, atomic force microscope infrared (AFM-IR)spectroscopy, and transient absorption microscopy. Among these methods,infrared-based imaging approaches are particularly attractive becausethey can extract molecular-specific information noninvasively and havemuch larger cross-section, when compared with Raman scattering. Yet,chemical imaging by conventional FTIR is hampered by the intrinsicallylow spatial resolution on the micron scale. AFM-IR provides nanoscaleresolution but is only applicable to extremely flat specimens underambient conditions. According to the present disclosure, a contact-free,easy to operate, and highly sensitive method for chemical imaging isprovided.

Recently developed mid-infrared photothermal (MIP) microscopy greatlyimproves According to the technology of the present disclosure, thelimitations of conventional MIP microscopy are overcome by afluorescence-enhanced mid-infrared photothermal (FE-MIP, also referredto herein as “F-MIP”) microscope. In this system and method of thedisclosure, a sample is labeled with a thermos-sensitive dye and themodulation of fluorescence intensity upon pulsed infrared excitation isprobed by a PMT.

FIG. 1A includes a schematic diagram illustrating operation of afluorescence-enhanced mid-infrared photothermal microscope 100,according to some exemplary embodiments. Referring to FIG. 1A, a sample102 being analyzed includes a fluorescent dye, which, in some particularexemplary embodiments, can be Rhodamine B. A pulse modulatedmid-infrared “pump” beam 104 impinges on sample 102. The infrared pulsescause an increase in temperature in the material of sample 102 and acorresponding increase in fluorescence in proximity to the regions ofsample 102 illuminated by the infrared pulses. That is, the temperatureincrease due to infrared absorption changes the molecular structure ofthe Rhodamine B fluorescent dye and thus change the intensity of thefluorescence induced when sample 102 is illuminated by a continuous wave(CW) illumination beam 108 of visible light. Hence, fluorescenceemission 106, induced by CW illumination beam 108 is modulated by pulsedinfrared beam 104. This modulation of the fluorescence is detected andmeasured such that the modulated temperature change in sample 102 can beanalyzed and associated with the sample, such as producing an image ofthe sample with extremely high resolution and sensitivity. Thus, theFE-MIP of the present disclosure not only takes advantage of the highspatial resolution from MIP, but also harnesses the high sensitivity ofcertain fluorescent dyes to temperature. The fluorescence label furtherenables the technology of the disclosure to probe the vibrationalspectrum of specific organelles inside a cell.

In some exemplary embodiments, the mid-infrared beam is apulse-modulated beam. A pulse repetition frequency of pulses in themid-infrared beam can be in a range of 1.0 to 1,000 kHz and cannominally be 100 kHz. An on-time of a pulse of the mid-infrared beam canbe in a range of 1.0 nanosecond to 1.0 millisecond and in someembodiments can nominally be between 50 and 1000 nanoseconds. A dutycycle of the mid-infrared beam can be in a range of 0.01% to 50% and insome embodiments can be between 1 and 10%.

When considering fluorescence as the reporter of temperature rise causedby mid-infrared absorption, according to the present disclosure, thefluorescent molecular thermometers should simultaneously satisfy certaincharacteristics, namely, high thermal sensitivity, high fluorescenceintensity, and robustness on excitation. Such dyes include, for example,rhodamine, fluorescein, cy2, cy3, Nile red, and green fluorescentproteins. In some exemplary embodiments, as noted above, Rhodamine 6Gand Rhodamine B are chosen as the fluorescent molecular thermometers,since they exhibit these preferred characteristics. Rhodamine dyes arexanthene derivatives presenting photo-physical properties well suitedfor a wide range of applications. Rhodamine and its derivatives havelong been known for the sensitivity of their fluorescence totemperature. Rhodamine B's fluorescence quantum yield drops withincreasing temperature as a consequence of the rotation of diethylaminogroups on the xanthene ring, as shown in FIG. 1B, which is a schematicdiagram illustrating molecular structure change of Rhodamine B caused byheating. Specifically, FIG. 1B illustrates rotation of diethylaminogroups on the xanthene ring of Rhodamine B with changing temperature.The Rhodamine B will change from the zwitterion form of Rhodamine B (RhBZ) to the lactone form RhB L during heating and vice versa.

According to the technology of the present disclosure, using thefluorescence dye as a probe to measure the photothermal effect by themid-infrared pump can dramatically increase the sensitivity of MIPimaging. The MIP signal contrast is conventionally attributed totemperature-induced changes of local refractive index. The thermallyinduced refractive index changes in conventional systems led totransient variations of an effective sample/medium light scatteringcross-section. Mie scattering theory is used to describe the relationbetween scattered light and the refractive index. There is a temperaturedependence of refractive index for most materials. Taking PMMA as anexample, the refractive index changes around 0.02% per Kelvin intemperature between 0 and 80° C. In MIP imaging, the temperatureincrease induced by the mid-infrared light is around 1 to 5 Kelvin.Thus, the scattering intensity is estimated to change around 0.1%percent per Kelvin. As a comparison, Rhodamine fluorescence intensityhas a much larger response to the temperature. Taking Rhodamine B forinstance, the fluorescence intensity drops around 2% per Kelvin, whichis two orders of magnitude larger than the scattering modulation depthcaused by refractive index change.

FE-MIP microscope 100 of the present disclosure provides another benefitfor chemical imaging, that is, the ability to record the vibrationalspectrum of a specific component in a complex environment. For example,a mammalian cell contains numerous spatially organized organelles. Theprocess of chemically imaging a specific organelle in a complex cellularsystem is challenging. Thus far, only lipid droplets that are denselypacked with C—H bonds have been heavily studied by coherent Ramanscattering microscopy. In contrast, fluorescent probes have the abilityto specifically label every component in a cell. For example, RhodaminePhalloidin can attach to the cytoskeleton. Therefore, FE-MIP microscope100 can obtain an IR spectrum that presents the cytoskeleton chemicalinformation. Collectively, these benefits of FE-MIP microscope 100provide a highly sensitive and selective chemical imaging ofnanoparticles, fingerprinting of bacteria, and vibrational imaging ofcytoskeleton in mammalian cells.

FIG. 2A includes a schematic diagram of a point scanning FE-MIPmicroscope 100A, according to some exemplary embodiments. FIG. 2B is aschematic diagram of the effects at the sample 102 being analyzed.Referring to FIGS. 2A and 2B, microscope 100A is used to analyze sample102, which includes a fluorescent dye, which, in some particularexemplary embodiments, can be Rhodamine B. A pulsed mid-infrared pumpbeam is provided by a quantum cascade laser (QCL) 110 to a reflectiveoptic 112 which reflects the mid-infrared pump beam through a calciumfluoride (CaF₂) optic 114 to a reflective objective 118, which focusesthe IR pump beam 107 onto controlled locations on sample 102. The IRbeam can have a wavenumber in the range of 100 to 4000 cm⁻¹. Part of themid-infrared pump beam is reflected by CaF₂ optic 114 to a mercurycadmium telluride (MCT) detector 116, which measures the beam. A CWprobe beam at a nominal wavelength of 532 nm is generated and output bysource 120 and passes through dichroic mirror 124 and is focused atsample 102 by water immersion objective 126, resulting in fluorescence105 at sample 102. Dichroic mirror 124 reflects the CW beam returningfrom sample 102 to photomultiplier tube (PMT) 130, which collects anddetects and measures the light, including the effects of the modulatedfluorescence from sample 102, as indicated at 109.

FIG. 2C includes a schematic functional block diagram illustratingcomponents for control and analysis in the FE-MIP microscope, accordingto some exemplary embodiments. Referring to FIG. 2C, the photothermalsignal, which includes the effects of the modulated fluorescence fromsample 102, is collected by PMT 130. The signals from PMT 130 areamplified by resonant amplifier 134 and detected by lock-in amplifier136. All of the system components, including IR laser (QCL) 110, lock-inamplifier 136, resonant amplifier 134, PMT 130, MCT 116 and positionstage 138 interface with processor/computer 132, which is used tocontrol positioning of the sample stage 138 and synchronization of IRpump modulation and illumination, as well as data acquisition andanalysis.

FIG. 2A illustrates a point scanning configuration of FE-MIP microscope100A. Continuing to refer to FIGS. 2A through 2C, a pulsed mid-IR pumpbeam, generated by tunable (from 1000 to 1886 cm−1) quantum cascadelaser (QCL) 110, which can be, for example, a MIRcat2400 QCL,manufactured and/or sold by Daylight Solutions, operating at 100 kHzrepetition rate, passes through a calcium fluoride (CaF₂) cover glassoptic 114 and then is focused onto sample 102 through a gold-coatingreflective objective lens 118 (for example, 52×; NA, 0.65; EdmundOptics, #66589). Continuous-wave probe laser 120 (for example, Cobolt,Samba 532 nm) beam is focused onto the same spot of sample 102 from theopposite side by a high NA refractive objective 126 (for example, 60×;NA, 1.2; water immersion; Olympus, UPlanSApo). The probe beam is alignedto be collinear to the mid-IR pump beam to ensure the overlap of the twofocus to achieve a good signal level. A scanning piezo stage (forexample, Mad City Labs, Nano-Bio 2200) with a maximum scanning speed of200 μs/pixel is used to scan sample 102. The fluorescence excited the byprobe laser 120 is then collected by the high NA refractive objective126 and reflected by dichroic mirror 124 (for example, DMSP550R, 550 nmcutoff, THorlabs). The fluorescence is then filtered by longpass filter128 (for example, FEL0550, 550 nm cutoff, THorlabs) and collected by PMT130 (for example, Hamamastsu H10721-110). In the imaging procedureaccording to the disclosure, sample 102 on a CaF₂ cover glass is firstlocalized by the fluorescence signal. Then, the pulsed infrared pump isturned on. The modulated scattering is collected by PMT 130, and the MIPsignal is extracted by lock-in amplifier 136, which detects/demodulatesthe signal to recover the IR modulation signal. Before reflectiveobjective 118, the infrared laser beam passes through a CaF₂ slip inoptic 114, and the reflection of the infrared laser is measured by MCTdetector 116 for normalization of IR power at each wavelength.

FIG. 3A includes a schematic diagram of a wide-field FE-MIP microscope100B, according to some exemplary embodiments. FIG. 3B includes aschematic functional block diagram illustrating components for controland analysis in the FE-MIP microscope 100B of FIG. 3A, according to someexemplary embodiments. Referring to FIGS. 3A and 3B, a laser 150, forexample, a 1040 nm full spectrum laser 150, is used as the CW excitationlight source. In some particular exemplary embodiments, laser 150 isdouble-frequency to 520 nm. This beam is chopped by acousto-opticmodulator (AOM) 162 to produce probe beam pulses. The excitation beam152 is focused on the back aperture of objective 154 to achieve epiillumination. Camera 164 is disposed to collect the fluorescence signalfiltered by long pass filter 166, which can be a 520 nm long passfilter. The mid-infrared pulse signal is generated by QCL 160 to heatsample 102 and is synchronized with the frame rate of camera 164. Hotframes and cold frames generated by camera 164 are recorded, hot framesbeing generated when the IR pump effects are present, and cold framesbeing generated with they are not present. The difference between thehot and cold frames is used to create a photothermal image.

Continuing to refer to FIGS. 3A and 3B, in some exemplary embodiments,function generator 170 can be used to trigger and synchronize systemcomponents, including generation and modulation of the pulsed IR signalby laser 150, the CW illumination signal generated by laser 150, anddetection and data acquisition by camera 164. Processor or computer 172extracts the FE-MIP signal and further processes images from camera 164.

In the particular exemplary embodiment illustrated in FIGS. 3A and 3B,the same mid-IR laser source 160 as is used in the embodiment of FIGS.2A-2C is used to IR-pump sample 102. The pulsed mid-IR pump beam,generated by tunable (for example, from 1000 to 1886 cm−1) quantumcascade laser (QCL) 160 (for example, QCL, Daylight Solutions,MlRcat-2400) operating at 100 kHz repetition rate is focused by aparabolic mirror to illuminate sample 102. A 520 nm femtosecond pulselaser 150 as probe is epi-illuminated on sample 102. The laser 150focused on the back aperture of objective 154 thus forms evenillumination on sample 102. The fluorescence beam is then filtered by550 nm long pass filter 166 and collected by camera 164 (for example,FLIR Grasshopper3 GS3-U3-32S4M). In some exemplary embodiments, camera164 is operated at a nominal frame rate of 100 Hz. Half of the totalframes are tuned off, and by subtracting the IR-on frame from the IR-offframe, the fluorescence-coded MIP image is obtained.

According to some exemplary embodiments, to prepare bacterial samplesfor FE-MIP imaging, S. aureus ATCC 6538 can be used as a model strain.Living S. aureus cells are first fixed by 10% formalin solution (ThermoFisher Scientific), and then centrifuged and washed withphosphate-buffered saline solution. Triton-X solution (Sigma) can beadded to permeabilize the cell membrane to facilitate fluorescencestaining. Fluorescent labeling can be carried out by incubatingbacterial cells with 10⁻⁴ M Rhodamine 6G (Sigma) solution for one hourat room temperature in the dark. After final conjugation and washingsteps, bacterial cells can be deposited and dried on a CaF₂ coverslipfor imaging.

According to the present disclosure, the spectral fidelity of the FE-MIPsystem of the disclosure to measure the mid-infrared spectrum ofstandard samples is described. As described above, Rhodamine 6G indimethyl sulfoxide (DMSO) and aqueous solutions as standard samples arechosen. The point scanning embodiment of FIGS. 2A-2C are used, and theFE-MIP spectrum is measured with 0.02 mW excitation power and the mid IRtuning from 1000 to 1750 cm−1. The spectrum scanning speed is 50 cm−1/s.The spectral fidelity is confirmed by comparing the MIP spectra to theFourier Transform infrared (FTIR) spectra, as shown in FIG. 4A. In FIG.4A, the FE-MIP spectrum of DMSO is illustrated. The FE-MIP spectrumshows 10 μMol/L R6G DMSO solution with spectrum scanning speed 50 cm−1.The FE-MIP spectrum for water is shown in FIG. 4B. The FE-MIP spectrumof single 200 nm RhB PS beads, spectrum scanning speed 50 cm−1. FIGS. 4Cand 4D illustrate the fluorescence and FE-MIP images of 200 nm beads.Pixel dwell time: 1 ms. Step size: 100 nm; Scale bar: 1 μm.

The sensitivity of FE-MIP is confirmed by mapping 200 nm polystyrene(PS) beads labeled with Rhodamine B dye. The beads are dispersed on acalcium fluoride coverslip. FIGS. 4C and 4D illustrate the fluorescenceand FE-MIP images of PS beads with the IR laser tuned to 1450 cm⁻¹. Tovalidate the spectral fidelity, an individual bead is analyzed, and theFE-MIP spectrum is recorded in the fingerprint region. The spectrum isconsistent with the FTIR spectrum. The scattering-based MIP and FE-MIPare consistent. Yet, under the same power of 16 mW for the IR laser, 30mW in the probe was used for the scattering-based MIP. In contrast, only0.02 mW of probe was used for recording the FE-MIP image.

When imaging bio-samples, especially living cells, the phototoxicitymust be taken into consideration. Compared with the scattering method,the FE-MIP of the current disclosure is very frugal in photo budget. Forthe FE-MIP method, the power density for the probe beam is around 1KW/cm{circumflex over ( )}2. While, the scattering MIP uses a probepower density 100 kW/cm², which is higher than FE-MIP by 2-3 orders ofmagnitude.

The FE-MIP microscopy of the present disclosure is applicable tochemical imaging of bacteria. According to some exemplary embodiments,S. aureus was imaged using the point scanning approach according to theembodiments described in detail above in connection with FIGS. 2A-2C.The images obtained are illustrated in FIGS. 5A through 5D. FIG. 5A isthe fluorescence image obtained for S. aureus bacteria, according tosome exemplary embodiments. FIGS. 5B-5D are FE-MIP images of S. aureusat the 1650 cm⁻¹, 1080 cm⁻¹, and 1750 cm⁻¹ IR pump laser wave numbers,corresponding to DNA, protein and lipid, respectively. FIG. 5E is acurve illustrating the FE-MIP spectrum and scattering MIP spectrum forS. aureus. Due to the sensitivity property of the FE-MIP of the presentdisclosure, far smaller probe intensity is used to image the spectrum ofbacteria, which can minimize the phototoxicity.

The performance of wide field FE-MIP for bacterial imaging, according tothe present disclosure, is also verified, as shown in FIGS. 6A through6D. According to some exemplary embodiments, S. aureus was also imagedusing the wide field approach according to the embodiments described indetail above in connection with FIGS. 3A-3B. The images obtained areillustrated in FIGS. 6A through 6D. FIG. 6A is the fluorescence imageobtained for S. aureus bacteria, according to some exemplaryembodiments. FIGS. 6B-6D are FE-MIP images of S. aureus at the 1650cm⁻¹, 1080 cm⁻¹, and 1750 cm⁻¹ IR pump laser wave numbers, correspondingto protein amide I band, nucleic acid phosphate band, and off-resonance,respectively.

According to the present disclosure, fluorescence imaging is asophisticated technique that is applied to FE-MIP to provide importantimprovements. For example, the illumination microscope can effectivelyimprove the resolution by two times and can be directly applies in thewide field system and realize super resolution chemical imaging. Othersuper resolution microscope methods, including, for example, stimulatedemission depletion (STED) microscope methods can also be applied to thetechnology described herein.

As described herein, the FE-MIP microscopy of the present disclosure canbe applied to take the fingerprint spectrum of the cytoskeleton, whichis buried inside the cell. By fluorescent labelling, the cytoskeletoncan be located, and its spectrum can be measured. It is noted that thetechnology is not only applicable to the cytoskeleton. For example,Rhodamine 123 has the ability to label mitochondrion. The greenfluorescent protein, which is known for labeling cells, is alsothermally sensitive. Thus, the green fluorescence can be appliedaccording to the disclosure to image the chemical information.

Mid-infrared photothermal microscopy according to the present disclosureis a chemical imaging technology in which a visible beam senses thephotothermal effect induced by a pulsed infrared laser. This technologyprovides infrared spectroscopic information at sub-micron spatialresolution and enables infrared spectroscopy and imaging of living cellsand organisms. However, current mid-infrared photothermal imagingsensitivity suffers from a weak dependance of scattering on temperature,and the image quality is vulnerable to the speckles caused byscattering. The present disclosure is directed to a novel version ofmid-infrared photothermal microscopy in which thermo-sensitivefluorescent probes are used to sense the mid-infrared photothermaleffect. According to exemplary embodiments, the fluorescence intensitycan be modulated at the level of 1% per Kelvin, which is 100 timeslarger than the modulation of scattering intensity. In addition,fluorescence emission is free of interference, thus greatly improvingthe image quality. Moreover, fluorophores can target specific organellesor biomolecules, thus augmenting the specificity of photothermalimaging. Spectral fidelity is confirmed through fingerprinting a singlebacterium. Finally, the photobleaching issue, in which the fluorophoremolecules are damaged by the visible light, is successfully addressedthrough the development of the wide-field fluorescence-detectedmid-infrared photothermal microscope which allows video ratebond-selective imaging of biological specimens.

Visualizing the molecular composition and monitoring the moleculardynamics in a complex living system is a central theme of life science.Fluorescence microscopy has been widely adopted in biomedical researchas it provides high-speed background-free imaging with exquisitemolecular specificity and superior resolution reaching the nanometerscale. While fluorescence microscopy excels at mapping the distributionand dynamics of tagged organelles such as mitochondria and biomoleculessuch as glucose and cholesterol, it does not provide chemicalinformation of the tagged cells or organelles. Lacking such informationhinders functional analysis, such as assessment of cell metabolicactivity.

Providing chemical specificity, high-speed and high-sensitivityvibrational spectroscopic imaging is an emerging platform.Recently-developed coherent Raman scattering microscopy, based oncoherent anti-Stokes Raman scattering (CARS) or stimulated Ramanscattering (SRS), has allowed real-time vibrational imaging ofbiomolecules in living cells and tissues. Advanced instrumentation haspushed the stimulated Raman spectral acquisition speed to microsecondscale. Adoption of stable isotope probes and alkyne-based Raman tagsgreatly enhanced the detection sensitivity, specificity andfunctionality in SRS microscopy. Being highly sensitive to C—Hvibrations, CARS and SRS imaging have unveiled new signatures of lipidmetabolism in a variety of biological systems. In comparison, high-speedCARS or SRS imaging of fingerprint Raman bands remains difficult.

Mid-infrared spectroscopy is complementary to Raman spectroscopy. UnlikeRaman scattering, the infrared absorption cross section in thefingerprint region is larger than in the high-wavenumber C—H vibrationalregion. Fourier transform infrared (FTIR) spectroscopy is one of themost extensively used techniques for chemical characterization andanalysis of biological cells and tissues The inherent vibrationabsorption of mid-infrared photons by biological macromoleculesincluding proteins, lipids, carbohydrates, and nucleic acids showsdistinctive absorption bands. Shifts in relative heights of absorptionbands, peak positions, and peak shape provide rich biomolecularinformation, including concentration, conformation, and orientation.FTIR spectroscopy has provided new insights in tissue classificationdrug and tissue interaction, neurodegenerative diseases, cancerprogression, and so on. However, the spatial resolution of infraredspectroscopic imaging is limited by the long mid-infrared illuminationwavelength, ranging from 5 to 20 μm. Strong water absorption furtherhinders its application to living cells.

To overcome these limitations in infrared spectroscopy, a new platform,termed mid-infrared photothermal (MIP) microscopy, has been developed toreach sub-micron spatial resolution. The MIP effect relies on aphotothermal process in which infrared absorption corresponds to aspecific molecular vibrational bond causes a localized temperature riseat the vicinity of target molecules. This photothermal effectconsequently induces a change of refractive index and a thermalexpansion. The MIP signal is then obtained by probing these changesusing a visible beam which provides a much smaller diffraction limitthan the mid-infrared illumination, enabling a spatial resolution downto 300 nm. Following the first demonstration of MIP imaging of livingcells, technical innovations have been made to enable MIP detection inwide-field, using scattering or phase signals. Meanwhile, MIP microscopyand its commercial product have found various applications in studyingliving cell, pharmaceuticals, viruses, and bacteria. Yet, thephotothermal effect induces only a tiny change in intensity and angulardistribution of the scattered probe light, due to the weak thermaldependence of particle size and refractive index. Typical fractionalchange is on the order of 10⁻⁴/K, set by the intrinsic thermalproperties of most materials. Such small modulation depth limits thesignal to noise ratio (SNR), especially in the wide-field mode where theprobe beam intensity at each pixel is limited by the well depth of aCMOS camera.

According to the present disclosure, a fluorescence-detected MIP (F-MIP,also referred to herein as “FE-MIP”) microscope, for example, microscope100A of FIG. 2A and microscope 100B in FIG. 3A, that utilizesthermo-sensitive fluorescent dyes as probes of the photothermal effectis provided. The F-MIP principle is illustrated in FIG. 7A. Referring toFIGS. 1A and 7A, a sample 102 stained with a thermo-sensitive dye isheated upon IR absorption by targeted molecules. The infrared pulsetrain 104 heats the surroundings of the fluorescent probe and causes atemperature rise, which subsequently modulates the fluorescence emissionefficiency. Such modulation is then measured by a lock-in amplifier,such as, for example, lock-in amplifier 136 illustrated in FIG. 2C.Using fluorescent dye to measure the temperature has been known. Yet, ithas not been used as a probe for infrared spectroscopic imaging. Thepresent technology provides important advantages over scattering-basedMIP microscopy. One advantage is the utilization of much largerphotothermal response of fluorescent dyes compared to scattering. Commonfluorophores including FITC, Cy2, Cy3, Rhodamine, and green fluorescentprotein have temperature-dependent emission efficiency on the order of1%/K, which is nearly 100 times larger than scattering dependence ontemperature. Thus, one can in principle boost the mid-infraredphotothermal imaging speed by two orders of magnitude. As a secondadvantage, as fluorescence appears at a new wavelength from the incidentbeam, it is insensitive to the laser relative intensity noise. As athird advantage, unlike scattering, fluorescence is incoherent and thusdoes not generate interference patterns. As a fourth advantage,fluorescent probes can target specific cells, intracellular organelles,or specific molecules, thus offering an enhanced specificity beyond thereach by scattering-based MIP microscopy. In the present disclosure, twoF-MIP systems are provided, one in a point-scanning mode, illustrated assystem 100A in FIG. 2A, and one in a wide-field mode, as illustrated assystem 100B in FIG. 3A, as described in detail herein.

Experimental Section

For an experiment with scanning F-MIP microscope 100A: A pulsed mid-IRpump beam is generated by a tunable (from 1000 to 1886 cm⁻¹) quantumcascade laser (QCL, Daylight Solutions, MlRcat-2400) operating at 100kHz repetition rate and 900 ns pulse duration. The IR beam passesthrough a calcium fluoride (CaF₂) cover glass and is then focused onto asample through a gold-coating reflective objective lens (52×; numericalaperture (NA), 0.65; Edmund Optics, #66589). A continuous-wave probelaser (Cobolt, Samba) at 532 nm is focused onto the same spot from theopposite side by a refractive objective (60×; NA, 1.2; water immersion;Olympus, UPlanSApo). The probe beam is aligned to be collinear to themid-IR pump beam. The reflective objective is fine tuned in 3D to ensureoverlap of the two foci. A scanning piezo stage (Mad City Labs, Nano-Bio2200) with a maximum scanning speed of 200 μs/pixel is used to scan thesample. The fluorescence is collected by the same refractive objective,reflected by a dichroic mirror (Thorlabs, DMSP550R, 550 nm cutoff),filtered by a long-pass filter (Thorlabs, FEL0550, 550 nm cut-off), andthen collected by a PMT (Hamamatsu, H10721-110). Specimens on a CaF₂coverglass are first imaged by fluorescence. Then, the pulsed IR laseris turned on and the modulated fluorescence signal is collected by thesame PMT. The F-MIP signal is extracted by a lock-in amplifier (ZurichInstruments, HF2LI). A laboratory-built resonant circuit is used toamplify the photocurrent from the PMT before it is sent to the lock-in.Before the reflective objective, the infrared laser passes through theCaF₂ slip and the reflected infrared laser intensity is measured by amercury cadmium telluride (MCT) detector for normalization of IR powerat each wavelength.

For an experiment with wide-field F-MIP microscope 100B: The IR pulsesare generated by the same QCL used in scanning F-MIP. The visible probebeam for fluorescence excitation (wavelength at 488 nm or 520 nm) isobtained by second-harmonic generation of a quasi-continuous femtosecondlaser tuned to 976 nm or 1040 nm (Coherent Inc, Chameleon, 140 fs, 80MHz). Prior to second-harmonic generation, the femtosecond beam ischopped into a 200-kHz pulse train (300 ns pulse width) by anacousto-optical modulator (AOM, Gooch and Housego). The IR beam passesthrough the substrate and is weakly focused onto a sample by a parabolicmirror (f=15 mm, Thorlabs, MPD00M9-M01). Using a Kohler illuminationconfiguration, the probe beam is focused on the back focal plane of theobjective lens (50×, 0.8 NA, Nikon) by a condenser (f=75 mm,AC254-075-A, Thorlabs). The fluorescence emission is collected by thesame objective lens, and after a long pass filter, collected by a CMOScamera (FLIR, Grasshopper3 GS3-U3-51S5M). The F-MIP images are acquiredby a virtual lock-in camera approach. Briefly, a pulse generator(Emerald Pulse Generator, 9254-TZ50-US, Quantum Composers) generates amaster clock signal at 200 kHz and externally triggers the QCL, the AOMand the CMOS camera to synchronize the IR pump pulses, the probe pulses,and camera exposure. The schematic is shown in FIG. 3A.

Cancer cell culture and staining: Mia Paca2 cells were purchased fromthe American Type Culture Collection (ATCC). The cells were cultured inRPMI 1640 medium supplemented with 10% FBS and 1% P/S. All cells weremaintained at 37° C. in a humidified incubator with 5% CO₂ supply. ForNile red staining, cells were incubated with 10 μM Nile Red (Invitrogen)for 30 minutes at room temperature followed with 15 minutes fixation in10% neutral buffered formalin. For rhodamine 123 (Invitrogen) staining,cells were incubated with 10 μg/ml rhodamine 123 for 30 minutes at 37°C.

Bacterial culture and staining: Staphylococcus aureus (S. aureus) wasincubated in a MHB medium for 10 h. After centrifuging and washing inphosphate-buffered saline (PBS), the bacteria were fixed by formalinsolution for 0.5 h. Rhodamine 6G or Cy2 at 10⁻⁴ M was then added intothe bacteria pellet. The pellet was then resuspended and incubated for 1h. With final washing steps, 2 μL sample were dried on a CaF₂ coverslipfor imaging. Shigella flexneri expressing GFP was grown overnight at 37°C. on a tryptic soy agar plate. Colonies with green fluorescence werepicked up by sterile inoculation loops and then resuspended in PBS. Thebacterial solution was diluted by optical density at 600 nm (OD600) to0.1. The bacteria were then fixed by 10% Formalin for 30 min at roomtemperature. The bacteria solution was washed twice by PBS beforeimaging.

FIG. 7A illustrates the fluorescence-detected mid-infrared photothermal(F-MIP) sensing approach, using point scanning microscope 100A, andspectral fidelity. Referring to FIG. 7A, modulation of fluorophoreemission intensity by pulsed infrared pump of surrounding molecules isillustrated. As described above in detail FIG. 2A illustrates theschematic of point scanning F-MIP microscope 100A. A pulsed mid-IR pumpbeam from a quantum cascade laser (QCL) and a continuous visiblefluorescence excitation beam are focused at the sample with a reflectiveobjective and a water-immersion objective, respectively. Thefluorescence emission is reflected by a dichroic mirror (DM), filteredand directed to a photomultiplier tube (PMT). A beam splitter is placedto reflect the scattered visible beam to a photodiode forscattering-based MIP (Sc-MIP) imaging. FIG. 2C illustrates theelectronics connections. The photothermal signal is detected by a PMTconnected to a resonant amplifier (RA) and detected by a lock-inamplifier. A PC is used for controlling the scanning stage and dataacquisition. FIG. 7B illustrates F-MIP spectra of DMSO supplemented withvarious thermo-sensitive fluorescent dyes. For each dye, standarddeviation of three independent measurements is shown at each wavenumber.The FTIR spectrum of DMSO (black) is shown for comparison.

Results and Discussion

Point-Scanning F-MIP Microscope 100A and Spectral Fidelity

Based on the principle illustrated in FIG. 7A, we have built a scanningF-MIP microscope as shown in FIG. 2B. A QCL laser provides IR pulsestunable in the entire fingerprint region. The repetition rate and pulsewidth were set to be 100 kHz and 900 nanoseconds, respectively. Two CWlasers at 532 nm and 488 nm were used for fluorescence excitation. Thefluorescence was detected by a photomultiplier tube and the F-MIP signalwas extracted by a lock-in amplifier. On the same setup, a photodiodewas also installed for scattering-based MIP (Sc-MIP) imaging using the532-nm laser as the probe beam. The electronics connections are shown inFIG. 2C.

Using this system, we have validated the spectral fidelity of F-MIPmicroscope 100A. We dissolved various thermo-sensitive dyes in DMSO, at100 μM concentration. We then recorded the F-MIP signals while scanningthe QCL laser. At each wavenumber, the F-MIP intensity was normalized bythe IR intensity measured by MCT. In all cases (FIG. 7B), the F-MIPspectra show the same peak intensity and width as the FITR spectrum ofDMSO. Importantly, because the dye concentration (100 μM) is much lowerthan DMSO concentration (14 M), the dyes do not interfere with the F-MIPspectra. These data demonstrate the F-MIP microscope is able to producereliable spectral information.

FIGS. 8A through 8G illustrate F-MIP imaging and fingerprinting singleS. aureus. FIG. 8A illustrates fluorescence image of S. aureus stainedwith R6G. Scale bar: 1 μm. Pixel dwell time: 1 ms. Fluorescenceexcitation: 0.025 mW at 532 nm. FIG. 8B illustrates F-MIP image of sameS. aureus at 1650 cm⁻¹. FIG. 8C illustrates fluorescence image of S.aureus stained with Cy2. Fluorescence excitation: 0.025 mW at 488 nm.FIG. 8D illustrates F-MIP image of same S. aureus at 1650 cm⁻¹. FIG. 8Eillustrates scattering image of S. aureus. Pixel dwell time: 1 ms.Visible probe power: 1.5 mW. FIG. 8F illustrates Sc-MIP image of same S.aureus at 1650 cm⁻¹. FIG. 8G illustrates the fingerprint spectra of S.aureus measured by F-MIP and Sc-MIP, respectively. For both F-MIP andSc-MIP, the IR laser power 1650 cm⁻¹ was 10.2 mW at sample. The FTIRspectrum recorded from a film of dried S. aureus was adopted from Li,X.; Zhang, D.; Bai, Y.; Wang, W.; Liang, J.; Cheng, J.-X.,“Fingerprinting a living cell by Raman integrated mid-infraredphotothermal microscopy,” Analytical Chemistry 2019, 91 (16),10750-10756.

F-MIP Imaging and Fingerprinting of Single Bacteria

We applied the F-MIP microscope 100A to image single S. aureus bacteriato evaluate its chemical imaging capability on biological specimens(FIGS. 8A-8D). The S. aureus culture was diluted to a concentration ofaround 5×10⁵/mL and then dried on a CaF₂ substrate. The S. aureusparticles were stained with fluorescence dye Cy2 and R6G, respectively.For each specimen, we acquired fluorescence and F-MIP images of the samebacteria at 1650 cm⁻¹ targeting the amide I band. For the F-MIP images,SNRs of 26 and 34 were achieved for R6G and Cy2 labeled bacteria at thefluorescence excitation power of 0.025 mW at the sample. For comparison,we recorded scattering and MIP images of the same specimen (FIGS.8E-8F). In order to obtain a similar SNR of 37, an excitation power of1.5 mW at sample was required, which is 60 times of the probe power usedfor F-MIP. On a single bacterium, we recorded the vibrationalfingerprint spectrum (FIG. 8G). The F-MIP spectra based on R6G and Cy2matches well the FTIR spectrum, showing distinct peaks at 1650, 1550,and 1080 cm⁻¹ for protein amide I, protein amide II, and nuclei acidphosphate vibrations, respectively. This comparison demonstrates thespectral fidelity of F-MIP in single bacterium analysis. Notably, thescattering and the Sc-MIP image both show a ring structure, with brightcontrast from the peripheral of the cell (FIGS. 8E-8F), whereas theF-MIP images (FIGS. 8B, 8D) show bright contrast from the entire cell.This result indicates that F-MIP is immune to edge-enhanced backscattering from a sizable particle.

FIGS. 9A though 9I illustrate Sc-MIP and F-MIP images of living MiaPaca2cancer cells. FIG. 9A illustrates scattering image of a MiaPaca2 cell.FIG. 9B illustrates Sc-MIP image of the same cell at 1650 cm⁻¹. FIG. 9Cillustrates Sc-MIP image at 1750 cm⁻¹. FIG. 9D illustrates afluorescence image of the same MiaPaca2 cell stained with Nile red. Theprobe laser is at 532 nm. FIG. 9E illustrates F-MIP image at 1650 cm⁻¹.FIG. 9F illustrates F-MIP image at 1750 cm⁻¹. FIG. 9G illustratesfluorescence image of a different MiaPaca2 cell stained with Rhodamine123 (Rho123). The probe laser is at 488 nm. FIG. 9H illustrates F-MIPimage at 1650 cm⁻¹. FIG. 9I illustrates F-MIP image at 1750 cm⁻¹. Scalebar: 5 μm.

Sc-MIP and F-MIP Imaging of Cancer Cells

Compared to bacteria, eukaryotic cells contain a nucleus and highlyorganized organelles in the cytoplasm. In the transmission image shownin FIG. 9A, the scattering-based contrast shows the overall cellmorphology. Accordingly, the Sc-MIP image (FIG. 9B) at 1650 cm⁻¹corresponding to the amide I band shows the protein content inside thenucleus and protein-rich structures in the cytoplasm, withoutspecificity to a certain organelle. The contrast decreases when the IRlaser is tuned to 1750 cm⁻¹ (FIG. 9C), showing the chemical specificity.In contrast, fluorescence microscopy is able to visualize specificbiomolecules and/or intracellular organelles via the versatilefluorescent probes. For instance, Nile red can selectively stain theintracellular lipid droplets and membranes while Rhodamine 123 is aspecific probe for localizing mitochondria in living cells. By stainingthe same cell with Nile red and excitation of the dye at 532 nm,phospholipid membranes in the cell are visualized (FIG. 9D), where thebrightest contrast is likely from the ER membrane. The F-MIP image at1650 cm⁻¹ gives the distribution of proteins in areas labeled by Nilered (FIG. 9E). The contrast nearly disappears when the IR laser is tunedto 1750 cm⁻¹ (FIG. 9F), showing the chemical specificity. To show thatour method is applicable to other dyes, we performed an independentexperiment in which living MiaPaca2 cells were labeled by Rhodamine 123targeting intracellular mitochondria (FIG. 9G). In accordance, the F-MIPimage at 1650 cm⁻¹ shows selective and bright contrast from theRhodamine 123 labeled region (FIG. 9H) and the contrast disappears at1750 cm⁻¹ (FIG. 9I).

From Point-Scanning to Wide-Field F-MIP

In the above experiments, the power used for F-MIP imaging is at themicrowatt level and is 60 times less than the power used for scatteringMIP imaging. The extremely low photon budget for F-MIP imaging opens theopportunity of increasing the throughput via wide-field illuminationwith IR pump pulses and visible probe pulses. Importantly, compared toscanning F-MIP, wide-field F-MIP significantly reduces the fluorophorephotobleaching rate based on the following consideration. In theexperiment, the IR pulse is 900 ns in duration and the pulse-to-pulseduration is 10 μs. In the aqueous environment, the temperature profilelargely follows the IR pulse. Thus, in a scanning experiment where acontinuous wave probe laser is used, the duty cycle is about 10%. Yet,in a wide field measurement, only two visible pulses are needed tomeasure the IR-on and IR-off states. Thus, the duty cycle can be 50%. Inthis way, the probe laser power can be significantly reduced, thusalleviating the photobleaching issue. Notably, our group recentlydemonstrated scattering-based wide-field MIP imaging. Yet, thesignal-to-noise ratio at the high-speed mode is limited by the weakdependence of scattering on temperature and the small well depth of theCMOS camera. As a result, a large number of integrations were needed toaccumulate sufficient photons to probe the MIP signal. Unlike thescattering photons, the fluorescence usually does not saturate thecamera. Due to the high thermo-sensitivity of fluorescent probes, it isanticipated that the MIP signal can be extracted from two sequentialframes (hot and cold) without further average.

Wide Field F-MIP Imaging System

As described above in detail, FIG. 3A illustrates the wide field F-MIPmicroscope 100B of exemplary embodiments. The infrared laser generatedby the QCL 160 is focused by a parabolic mirror. The visible lightmodulated by AOM 162 illuminates the sample and excites thefluorescence. The fluorescence is filtered and collected by CMOS camera164. FIG. 10A illustrates temporal synchronization of the IR pump pulse,fluorescence excitation pulse, and camera exposure. FIGS. 10B and 10Cillustrate a wide field fluorescence image of Cy2-stained S. aureus withIR on and IR off, designated as hot and cold, respectively. FIG. 10Dillustrates wide field F-MIP at 1650 cm⁻¹, and FIG. 10E illustratesF-MIP at 1400 cm⁻¹ off resonance. Scale bar: 10 μm. Fluorescenceexcitation: 488 nm, 1 mW. IR pulse rate: 200 KHz. Camera frame rate: 40Hz.

In wide-field F-MIP microscope 100B shown in FIG. 3A, the pulsedinfrared laser is weakly focused onto a sample by a parabolic mirror. An80-MHz femtosecond laser is modulated by an AOM and frequency-doubled tovisible window. The broad bandwidth of femtosecond pulses reducesspeckles in scattering-based MIP imaging. The virtual lock-in detectionscheme is illustrated in FIG. 10A. The fluorescence excitation pulse issynchronized with the IR pump pulse. In the cold frame, no IR pumppulses heat the sample. The camera detects the hot and cold framessequentially at an exemplary 40 Hz frame rate. The difference (cold−hot)generates the MIP image. The average probe laser power is approximately1 mW and the exposure time is approximately 300 ns. The IR power is inthe range from 5 to 15 mW depending on the spectral window examined. Tocharacterize the spatial resolution, we mapped Cy2-label polystyrenebeads with diameter of 500 nm, the F-MIP intensity profile shows afull-width at half maxim of 610 nm. After deconvolution with particlesize, the spatial resolution is estimated to be 390 nm, which is closeto the diffraction limit of the 0.8 NA objective.

To demonstrate the applicability of wide-field F-MIP to biologicalspecimens, we deposited S. aureus stained with Cy2 onto a siliconsubstrate and measured the fluorescence with IR on and IR offsequentially. The hot frame and the cold frame are illustrated in FIGS.10B and 10C, respectively. By subtracting the hot from the cold frame,the intensity difference generates the F-MIP image shown in FIG. 10D.When the IR laser is tuned to 1650 cm⁻¹, corresponding to the amide Iband of proteins, a signal to noise ratio of 30 was obtained. The IRpulse only heats the upper half of the field of view. For this reason,only the S. aureus particles in the upper part give the F-MIP contrast.When the IR laser is tuned to 1400 cm⁻¹, off resonance to major IRpeaks, the contrast nearly disappears, as illustrated in FIG. 10E. It isnoted that the wide-field F-MIP imaging speed of 20 frames per second is2000 times faster than the scanning F-MIP imaging speed (100 seconds perframe with a pixel dwell time of 1.0 millisecond).

Performance Comparison Between Wide-Field Sc-MIP and F-MIP

FIGS. 11A-11N illustrate performance comparison between wide fieldSC-MIP and wide field F-MIP systems. FIG. 11A illustrates a fluorescenceimage of S. aureus deposited on a silicon substrate. FIG. 11Billustrates a single frame F-MIP image of the same cells at 20 Hz speed.FIG. 11C illustrates F-MIP image of the same cells with 100 framesaverage. FIG. 11D illustrates a scattering image of S. aureus depositedon a silicon substrate. FIG. 11E illustrates a single frame Sc-MIP ofthe same cells shown in panel d at 20 Hz speed. FIG. 11F illustratesSc-MIP of the same field of view with 100 frames average. FIG. 11Gillustrates intensity profile along the white line marked in FIGS. 11Band 11E. FIG. 11H illustrates intensity profile along the white linemarked in FIGS. 11C and 11F. FIG. 11I illustrates fluorescence intensityof single S. aureus in sequentially acquired hot and cold frames. FIG.11J illustrates scattering intensity of single S. aureus in sequentiallyacquired hot and cold frames. The IR laser is tuned to 1650 cm⁻¹ fordata in FIGS. 11A-11K. FIG. 11K illustrates fluorescence image of Sheilaflexneri bacteria expressing GFP. FIG. 11L illustrates a single frameF-MIP of the same bacteria at 1650 cm⁻¹. FIG. 11M illustrates a F-MIPimage of the same field of view with 100 frames average. FIG. 11Nillustrates fluorescence intensity of the square area in sequentiallyacquired hot and cold frames.

The performance of fluorescence-enhanced and scattering-based MIPimaging in the wide field mode were compared, using S. aureus on asilicon substrate as testbed. FIGS. 11A-11C illustrate the fluorescenceand F-MIP images of individual S. aureus with the IR laser tuned to 1650cm⁻¹. The signal to noise ratio for a single bacterium reaches 33 insingle frame F-MIP and 275 after 100 frames average. FIGS. 11D-11Fillustrate the scattering and SC-MIP images of individual S. aureus withthe IR laser tuned to 1650 cm⁻¹. FIGS. 11G-11H illustrate the intensityprofile across the white line indicated in the images. The Sc-MIPcontrast is completely buried in the speckle pattern in singe frameacquisition. After 100 frames average, the signal to noise ratio reaches14, which is 20 times lower than F-MIP of the same sample. The muchhigher signal to noise ratio in F-MW can be attributed to the lack ofshot noise from scattered photons and the much larger thermo-sensitivityof the fluorescence probe. FIG. 11H shows similar line width for thebacterium in Sc-MIP and F-MIP. However, the F-MIP intensity does notsuffer from the interference (i.e. the dark ring around the peak)encountered in the Sc-MIP image. FIGS. 11I-11J illustrate the F-MIP andSc-MIP intensities from a single bacterium in sequentially acquired hotand cold frames. The modulation depth, defined as percentage ofintensity difference between hot and cold frames, is found to be about4% for F-MIP, but is buried in frame-to-frame fluctuations in Sc-MIP.Moreover, FIG. 11I shows negligible photobleaching in the recorded 50frames over a period of 1.25 seconds.

Besides fluorescent dyes, the feasibility of F-MIP imaging for cellsexpressing green fluorescent proteins (GFPs) was tested. It has beenshown that GFPs are highly thermo-sensitive with 1% intensity decreaseper degree in temperature rise. Accordingly, in F-MIP imaging of Sheilaflexneri bacteria expressing GFP (FIGS. 11K-11M), 2% fluorescenceintensity difference between cold and hot frames was observed (FIG.11N). The signal-to-noise ratio reaches 10 and 97 in single frame and100 frames average, respectively. The recorded 50 frames onlyexperienced 3% photobleaching. The fluorescence fluctuation from frameto frame was due to laser instability.

Photothermal microscopy is a pump-probe technique involving modulationof the pump beam and demodulation of the signal usually by a lock-in. Inthe present disclosure, the sensitivity is defined as the modulationdepth, ΔI_(pr)/I_(pr)=σI_(p), where σ is related to the thermalsensitivity, I_(p) is the IR pump, and I_(pr) is the probe intensity. Inthis sense, fluorescence-detected MIP is more sensitive thanscattering-based MIP due to the much larger thermo-sensitivity offluorophores. Experimentally, as shown in FIGS. 11A-11N, the modulationdepth in F-MIP is about 2%, whereas the modulation is buried inframe-to-frame intensity fluctuation in Sc-MIP.

We have defined the imaging SNR as the ratio of signal intensity fromsingle particles to pixel-to-pixel background fluctuations. Thus, inSc-MIP, the SNR=Signal/(Noise_PD+Noise_photon). In F-MIP,SNR=Signal/Noise_PMT. Under the shot noise limit, the photon noise isproportional to √{square root over (I_(pr))}. Based on this model, theSNR depends on the value of σ and the probe beam intensity if we assumethat same IR pump beam is used. In scanning MIP microscopy, the probepower (˜ 10 mW) in the scattering mode can be 10,000 times larger thanthat in the fluorescence mode (˜ 1 μW). In this case, the SNR in Sc-MIPis dominated by the photon noise, and the SNR=σ_(sc)I_(p)√{square rootover (I_(pr))}. In F-MIP, the SNR=σ_(f)I_(p)I_(pr)/Noise_PMT. In thiscase, the SNR in F-MIP is a trade-off between a much larger σ and a muchsmaller I_(pr) used. Experimentally, we observed similar SNR in F-MIPcompared to Sc-MIP (See FIGS. 8A-8G). However, in wide-field MIPmicroscopy, limited by the well depth of a common CMOS camera, the probepower at each pixel is at the nW level for both the scattering and thefluorescence modality. In this case, the detector noises dominate, andthe SNR in F-MIP can be theoretically larger by two orders of magnitudethan that in Sc-MIP. In the experiment, we showed that the SNR inwide-field MIP is 20 times larger than that in wide-field Sc-MIP.

Theoretically, F-MIP microscopy is based the thermal diffusion from thetarget molecule to the fluorescent probe. The thermal diffusion lengthis defined as μ_(t)=2√{square root over (αt)}, where α is the thermaldiffusivity. In an aqueous environment, the value of α is 1.4×10⁻⁷ m²/s.In our wide-field F-MIP experiment, the IR pulse is 900 ns in durationand the fluorescence excitation pulse is 300 nm. If we set thepump-probe delay to be 900 ns, the thermal diffusion length is ˜700 nm,which is slightly larger than the diffraction limit of the visible probebeam. If one can use IR pump and visible probe pulse of 5 ns durationand the pump-probe delay is set to be 5 ns, the thermal diffusion lengthcan be reduced to 50 nm. In this case, one can detect the chemicalcontent surrounding the fluorescence probe on the nanoscale. If theprobe is conjugated to the target molecule, like a GFP conjugated to aprotein, the intramolecular vibrational redistribution on the picosecondscale can be much faster than inter-molecular vibrationalredistribution. In principle, it could create an intramolecular F-MIPsignal, assuming that picosecond IR pump and probe pulses are used.

The dependence of thermo-sensitivity on two important environmentalfactors, salt concentration and viscosity, has been previously studied.It was shown that thermo-sensitivity is nearly independent of saltconcentration in the 10 to 100 mM range. Also, it was shown that PEGBODIPY lifetime in cells is due to temperature and independent ofchanges in viscosity. These data suggest that fluorophores'thermo-sensitivity can be used as a reliable readout of the mid-infraredphotothermal effect.

F-MIP microscopy opens new opportunities for live-cell chemical imaging.First, F-MIP greatly enhances the specificity of MIP microscopy. Toillustrate this advantage, one could normalize the F-MIP signal with thedirect fluorescence signal. As shown in FIGS. 12A-12F, the normalizedF-MIPs images exhibit a more uniform distribution of the signal arisingfrom proteins in regions labeled by the dyes. In contrast, individualmitochondria are clearly seen in the F-MIP image (FIG. 9H) due toenrichment of rhodamine 123 in the mitochondria. Second, by F-MIPspectroscopic imaging of specific organelles (e.g. lipid droplets)specifically labeled by a thermo-sensitive fluorophore (e.g. BODIPY),one will be able to tell not only the amount and distribution, but alsothe composition of lipids, which is beyond the reach by fluorescencemicroscopy alone. Such capacity will allow quantitation of lipidmetabolism in cells under various conditions (e.g., in response to astress). Third, F-MIP microscopy opens new ways to push the boundary ofmid-infrared photothermal microscopy. For example, integration ofinfrared laser excitation and light field fluorescence probing isexpected to enable single-shot volumetric infrared spectroscopic imagingat sub-micron spatial resolution. Additionally, structured illuminationcan be harnessed to break the diffraction limit of the visible beam,which is expected to push the spatial resolution of MIP microscopy to anew level.

Compared to MIP microscopy, F-MIP microscopy relies on labeling thespecimen with a thermo-sensitive dye and the signal level depends on thedye concentration. Unlike scattering-based MIP, F-MIP cannot detectvibrational excitation at locations where fluorescent dyes do not exist.When interpreting the F-MIP contrast, one should use the fluorescenceimage as a reference. For quantitative comparison of F-MIP signal levelbetween different particles, normalization with fluorescence intensityis needed. An alternative approach is to measure the thermal modulationof fluorescence lifetime instead of intensity. For the same reason, theSNR in F-MIP microscopy depends on the number of fluorophores in theparticles to be detected. For biological nanoparticles such as virionparticles, the small number of fluorescent labels may give a low signallevel and limit the SNR accordingly. In such case, detection ofinterferometric scattering becomes a more suitable approach. In fact,detection and fingerprinting of single virus particles has been achievedby an interferometric mid-infrared photothermal microscope.

Conclusions

In efforts to push the detection limit and increase the specificity ofoptically detected mid-infrared photothermal microscopy, a new platformtermed fluorescence-enhanced mid-infrared photothermal (F-MIP)microscopy is developed. Our platform harnesses thermo-sensitivefluorescent probes to sense surrounding temperature rise induced bypulsed infrared excitation. High spectral fidelity is demonstrated forfluorescent probes in DMSO solution and inside biological cells. In thepoint scanning modality, we have demonstrated F-MIP imaging andfingerprinting of a single bacterium. While using fluorescence as a readout, the fingerprint information would allow functional assessment ofbiological specimen, such as metabolic response of bacteria toantibiotics treatment. Furthermore, organelle-specific F-MIP imaging isachieved, which opens exciting opportunities of probing the chemicalcontent of intracellular organelles. In the wide-field modality, wedemonstrated video rate, high signal to noise ratio, speckle-free F-MIPimaging of individual bacteria. Finally, our platform is applicable tobiological cells expressing GFP. This approach opens new opportunitiesof monitoring secondary structure of specific proteins tagged by GFP,which is beyond the reach by IR spectroscopy or fluorescencespectroscopy alone.

Whereas many alterations and modifications of the disclosure will becomeapparent to a person of ordinary skill in the art after having read theforegoing description, it is to be understood that the particularembodiments shown and described by way of illustration are in no wayintended to be considered limiting. Further, the subject matter has beendescribed with reference to particular embodiments, but variationswithin the spirit and scope of the disclosure will occur to thoseskilled in the art. It is noted that the foregoing examples have beenprovided merely for the purpose of explanation and are in no way to beconstrued as limiting of the present disclosure.

While the present inventive concept has been particularly shown anddescribed with reference to exemplary embodiments thereof, it will beunderstood by those of ordinary skill in the art that various changes inform and details may be made therein without departing from the spiritand scope of the present inventive concept as defined by the followingclaims.

1. A system for microscopic analysis of a fluorescently labeled sample,comprising: a mid-infrared (IR) optical source for generating amid-infrared beam, the mid-infrared beam being directed onto at least aportion of the sample to induce a temperature change in the portion ofthe sample by absorption of the mid-infrared beam; an optical source forgenerating a probe beam, the probe beam being directed to impinge on thesample; a detector for detecting fluorescent emissions from the samplewhen the probe beam impinges on the sample; and a data acquisition andprocessing system for acquiring and processing the detected fluorescentemissions from the sample to: (i) generate a signal indicative ofinfrared absorption by the portion of the sample, (ii) generate a signalindicative of temperature in the portion of the sample based on thesignal indicative of infrared absorption by the portion of the sample,(iii) generate an image of the portion of the sample using the signalindicative of temperature in the portion of the sample.
 2. The system ofclaim 1, wherein the fluorescently labeled sample comprises a samplelabeled with a fluorescent dye comprising at least one of rhodamine B,fluorescein, cy2, cy3, Nile red and green fluorescent protein.
 3. Thesystem of claim 1, wherein the mid-infrared beam is a pulse-modulatedbeam.
 4. The system of claim 3, wherein a pulse repetition frequency ofpulses in the mid-infrared beam is in a range of 1.0 to 1,000 kHz. 5.The system of claim 3, wherein the pulse repetition frequency of pulsesin the mid-infrared beam is nominally 100 kHz.
 6. The system of claim 3,wherein an on-time of a pulse of the mid-infrared beam is in a range of1.0 nanosecond to 1.0 millisecond.
 7. The system of claim 3, wherein aduty cycle of the mid-infrared beam is in a range of 0.01% to 50%. 8.The system of claim 1, wherein the mid-infrared beam is scanned over aplurality of portions of the sample such that the image is generated fora plurality of portions of the sample.
 9. The system of claim 1,wherein: the mid-infrared beam is directed onto a plurality of portionsof the sample; and the detector comprises a two-dimensional array ofdetectors such that the image is generated for the plurality of portionsof the sample.
 10. The system of claim 9, wherein the detector comprisesa camera.
 11. The system of claim 1, further comprising an objectiveconfigured to focus the mid-infrared beam onto the sample.
 12. A methodfor microscopic analysis of a fluorescently labeled sample, comprising:directing a mid-infrared beam onto at least a portion of the sample toinduce a temperature change in the portion of the sample by absorptionof the mid-infrared beam; directing a probe beam to impinge on thesample; detecting fluorescent emissions from the sample when the probebeam impinges on the sample; and receiving and processing thefluorescent emissions from the sample, the processing including: (i)generating a signal indicative of infrared absorption by the portion ofthe sample, and (ii) generating an image of the portion of the sampleusing the signal indicative of infrared absorption by the portion of thesample.
 13. The method of claim 12, wherein the fluorescently labeledsample comprises a sample labeled with a fluorescent dye comprising atleast one of rhodamine B, fluorescein, cy2, cy3, Nile red and greenfluorescent protein.
 14. The method of claim 12, further comprisingpulse modulating the mid-infrared beam such that the mid-infrared beamis a pulse-modulated beam.
 15. The method of claim 14, wherein a pulserepetition frequency of pulses in the mid-infrared beam is in a range of1.0 to 1,000 kHz.
 16. The method of claim 14, wherein a pulse repetitionfrequency of pulses in the mid-infrared beam is nominally 100 kHz. 17.The method of claim 14, wherein an on-time of a pulse of themid-infrared beam is in a range of 1 nanosecond to 1 millisecond. 18.The method of claim 14, wherein a duty cycle of the mid-infrared beam isin a range of 0.01% to 50%.
 19. The method of claim 12, furthercomprising scanning the mid-infrared beam over a plurality of portionsof the sample such that the image is generated for a plurality ofportions of the sample.
 20. The method of claim 12, wherein: themid-infrared beam is directed onto a plurality of portions of thesample; and the detector comprises a two-dimensional array of detectorssuch that the image is generated for the plurality of portions of thesample.
 21. The method of claim 20, wherein the detector comprises acamera.
 22. The method of claim 12, further comprising focusing themid-infrared beam onto the sample with an objective.